chapter – 6 simultaneous determination of drug...

CHAPTER – 6

SIMULTANEOUS DETERMINATION OF DRUG RELEASE DURING DISSOLUTION

OF TELMISARTAN AND HYDROCHLOROTHIAZIDE IN TABLET

DOSAGE FORM USING REVERSE PHASE HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY

CHAPTER - 6

SPP SPTM, SVKM’s NMIMS, Mumbai 122

CHAPTER – 6

Simultaneous determination of drug release during dissolution of Telmisartan and Hydrochlorothiazide in tablet dosage form using reverse phase high performance liquid chromatography 6.0 INTRODUCTION The objective was to develop a single method for simultaneous determination of percentage

drug release of the actives in Telmisartan and Hydrochlorothiazide tablet dosage form. The

method was validated as per ICH guidelines Q2 (R1).

The target for this research work was to present comprehensive methods of critical tests for

each drug product. Thus this work can be treated as part of a monograph for the drug

product.

The main aim for this current study was to develop the analytical determination/ parameters

of drug release rather than the dissolution parameters.

A brief introduction of each molecule is has already been included in chapter 4.

6.1 LITERATURE SURVEY

The literature survey reveals that, TE and HCTZ are reported in British Pharmacopoeia [6, 7].

There have been several publications describing analytical methods for the determination of

HCTZ and TE individually or with other drugs as combination.

Although there are a few papers published on simultaneous determination of TE and HCTZ

in formulation most of them deal with the assay of each constituent. Several methods are

reported for the determination of TE like Spectrophotometric [7] and HPLC. [9-11] The other

methods available in the literature are based on Linear Sweep polarography, [12] LC–MS. [13]

Articles on the determination of HCTZ in combination with other drugs by HPLC are also

reported in literature. [14, 15]

However the exhaustive literature survey revealed that none of the most recognized

pharmacopoeias or any journals includes these drugs in combination for the simultaneous

determination of drug release of TE and HCTZ is not available@. So the aim of this work

was to develop a liquid chromatographic procedure which will serve a reliable, accurate,

sensitive HPLC method for the simultaneous determination of drug release of TE and HCTZ

in TE + HCTZ tablets.

------------------------------------------------------------------------------------------------------- @ This search was performed at the start and during the course of the study. A draft monograph has been published in

USP Forum Vol 36(3). The Monograph is now official as per USP 34, from December 2011. A comparison of

methods is given in the next section.

CHAPTER - 6


Table 6.1.1: Dissolution – Pharmacopeia methods for Drug Product

Telmisartan HCTZ Telmisartan-HCTZ

BP 2011 USP 34 BP 2011 USP 34 USP 35

Method Not Available UV

Spectrophotometer

Not Mentioned UV

Spectrophotometer

HPLC

Column Not

Applicable

Not Applicable Not Applicable Not Applicable C8

3mm x 6cm; 5µm

Column Temp Not

Applicable

Not Applicable Not Applicable Not Applicable 40°C

Mobile Phase Not

Applicable

Not Applicable Not Applicable Not Applicable Solution A: 5.0 g/L of ammonium

dihydrogen phosphate was prepared in

water. The pH was adjusted with

phosphoric acid to 3.0

Solution B: Acetonitrile

Gradient Not

Applicable

Not Applicable Not Applicable Not Applicable Time

(min)

%A %B

0 85 15

3.50 85 15

CHAPTER - 6


3.51 45 55

7.70 45 55

7.71 20 80

12.0 20 80

12.1 85 15

15.5 85 15

Flow Rate Not

Applicable

Not Applicable Not Applicable Not Applicable Flow gradient

Time

(min)

Flow

0 0.6

5 0.6

5.01 1

6.2 1

6.21 0.6

9.7 0.6

Wavelength Not

Applicable

296nm Not Applicable 270nm 270-Telmisartan

298- Hydrochlorothiazide

Inj Vol (µL) Not

Applicable

Not Applicable Not Applicable Not Applicable 4

CHAPTER – 6


6.2 PRESENT WORK AND DISCUSSION 6.2.1 Selection of Chromatographic Method Reverse Phase chromatography is the natural choice for method development because of

its ease of handling and robust nature. All development was conducted using reverse

phase methods. Official methods and methods published in literature for Telmisartan and

Hydrochlorothiazide are based on reverse phase chromatographic (RPC) separation.

Moreover since drug release is being studied in aqueous medium there is no option to go

for normal phase HPLC separations.

6.2.2 Selection of Stationary Phase

USP, EP monograph and reported HPLC methods of Telmisartan or hydrochlorothiazide

recommended use of C18 column for the purpose of determination of related impurities

and assay. So C18 columns were preferred as stationary phase. As described in the

previous chapter, C8 column was used in assay to reduce the run time to 15 minutes.

However for dissolution, 15 minutes is considered a long run time, especially when one

is considering dissolution profiles. Typically a six unit dissolution profile would involve

about 60 injections. Thus, a 15 minute run time for injection would result in an exercise

that would last for 15 hours which is not practical. Therefore, the shorter the run time,

the quicker the experiment can be planned for subsequent formulation development trial

batches. The usage of ion pairing agent was avoided since that would retain the

Telmisartan peak extensively. From the experience of the method development on C18

and C8 column, a highly polar Cyano (CN) column was preferred since the target was to

achieve respectable retention and separation of the two actives within an isocratic run.

The biggest challenge in using a cyano phase is the column to column variation. Thus

column quality and manufacturing reproducibility is of utmost importance. In this regard

ACE Cyano column was chosen for trials.

6.2.3 Selection of Wavelength for Analysis

The optimum wavelength selected was 270 nm which represents the wavelength where

both actives have suitable responses in order to permit simultaneous determination of

Telmisartan and HCTZ. 270nm is also the wavelength which has absorption maxima for

hydrochlorothiazide and absorption minima for Telmisartan. Thus, at this wavelength,

minor changes will not affect peak areas and consequently, the final result. This will, in

effect, produce a robust method with respect to wavelength.

CHAPTER – 6


Figure: 6.2.3.1 Overlaid UV spectrum of Telmisartan and Hydrochlorothiazide

6.2.4 Selection and Optimization of Mobile Phase Isocratic method was the target and it was achieved using the cyano column. Although

the dissolution method needs not be stability indicating, the known impurities

(essentially degradants from Hydrochlorothiazide) were separated from main peak. The

other concern was the peak shape since the injection will be of purely aqueous samples.

BCS solubility study was performed on each molecule. The results are given in Table 6.1

Table 6.1: BCS Solubility

Medium % dissolved

(HCTZ)

% dissolved

(Telmisartan)

Water 93.3 0.2

0.1 N HCl 85.1 100.2

0.01 N HCl 95.8 30.9

0.001 N HCl 95.0 0.7

Citrate pH 3.0 buffer 98.1 1.5

Acetate pH 4.5 buffer 97.8 0.1

Acetate pH 5.0 buffer 94.1 0.0

Phosphate pH 6.8 buffer 97.7 0.3

Phosphate pH 7.4 buffer 74.8 1.1

CHAPTER – 6


All the samples were analysed by the HPLC method and peak shapes were acceptable

proving that this method is capable to accept fully aqueous injections and thus suitable

for dissolution study.

Optimized Chromatographic Conditions:

Dissolution Conditions;

Instruments/Equipment : Dissolution Apparatus, Make-Electrolab, Model-TDT-

08L with fraction collector, or equivalent

Apparatus : USP Apparatus II (Paddle)

Dissolution medium : Phosphate Buffer pH 7.5

Volume : 900 ml

Temperature : 37°C ± 0.5°C

Speed : 75 rpm

Time : 60 minutes

Preparation of Phosphate Buffer pH 7.5:

Weigh accurately and transfer 6.8 gm of Potassium Dihydrogen Phosphate and 0.9 gm of Sodium

Hydroxide in 1000 ml of water. Adjust the pH 7.5 with Sodium Hydroxide solution.

The method employed for Telmisartan + Hydrochlorothiazide tablets is separation using isocratic

HPLC with detection by UV.

Chromatographic conditions:

Instruments/Equipment : HPLC, Make – Waters, Alliance, 2695 Separation

Module, (UV/PDA), or equivalent.

Analytical Balance, Make –Mettler Toledo, Model-

XS205DU, or equivalent.

Column : ACE Cyano, 150 x 4.6 mm, 5µm or equivalent

Flow rate : 2.0 ml/minute

Column temperature : 35°C

Wavelength : 270 nm

Sample temperature : 25°C

Injection volume : 20 l

Retention time : approx. 1.52 minutes for Hydrochlorothiazide

approx. 7.56 minutes for Telmisartan

CHAPTER – 6


Diluent:

0.1N Methanolic NaOH:

Weigh accurately and transfer 4 gm of Sodium hydroxide in 1000 ml volumetric flask add 40 ml

water sonnicate to dissolve and make up to mark with methanol.

Buffer:

Mix 2 ml of Triethylamine in 1000 ml water. Adjust pH 3.0 with ortho-phosphoric acid.

Preparation of Mobile Phase:

Prepare a mixture of Buffer: Acetonitrile: Methanol in the ratio 80:15:5 v/v/v. Mix and degas.

6.3 EXPERIMENTAL WORK

6.3.1 Instrumentation

Equipment Make Model

HPLC Waters 2695Alliance Separation

Module, (PDA/UV

Detector) 2996/2487

Column ACE ACE Cyano, 150 x 4.6

mm, 5µm

Dissolution

apparatus

Electrolab TDT-08L

TDT-14L

pH meter Thermo

Electron

Corp.

Orion-4star 1117000

Analytical

Balance

Mettler

Toledo

XS205DU

6.3.2 Chemicals and Reagents

Name Grade Manufacturer

Triethylamine HPLC grade Merck

Methanol Gradient grade Merck Acetonitrile Gradient grade Rankem

Sodium Hydroxide GR Merck Ortho-phosphoric acid GR Merck

Water HPLC milli-Q In-house

CHAPTER – 6


6.3.3 Working Standard

Working Standard:

Standard Lot .No. Potency (as is)

Telmisartan TE0010108 98.7

Hydrochlorothiazide HCT/60914 99.5

Test Sample:

Batch. No. Label claim (mg/tablets)

Telmisartan Hydrochlorothiazide

THT/80-12.5/034 80 12.5

THT/40-12.5/032 40 12.5

Placebo

Batch. No.

THT/80-12.5/034 P

THT/80-12.5/034 PH

THT/80-12.5/034 PT

6.3.4 Solution Preparation Preparation of Standard solution:

Standard Stock solution- Telmisartan:

Accurately weigh and transfer about 90.0 mg of Telmisartan standard a to a 50 ml volumetric

flask, add 30ml of diluent, sonnicate to dissolve and make up the volume with diluent.

Standard Stock solution- Hydrochlorothiazide:

Accurately weigh and transfer about 55.0 mg of Hydrochlorothiazide standard to a 100 ml

volumetric flask, add 70ml of Acetonitrile, sonnicate to dissolve and make up the volume with

Acetonitrile.

For 40-12.5 mg

Take 5 ml of Standard Stock solution of Telmisartan and 5 ml of Standard Stock solution of

Hydrochlorothiazide in 200 ml volumetric flask; make the volume with dissolution media.

CHAPTER – 6


For 80-12.5 mg

Take 10 ml of Standard Stock solution of Telmisartan and 5 ml of Standard Stock solution of

Hydrochlorothiazide in 200 ml volumetric flask; make the volume with dissolution media.

Preparation of Sample solution:

Accurately weigh one tablet and transfer in the dissolution vessel. Run the Dissolution as per

the set parameters. Withdraw about 10 ml of the sample after 60 minutes. Filter through on

line SS filter, discarding first 2-3 ml of filtrate inject the filtrate into the HPLC.

Preparation of Placebo solution:

Weigh accurately placebo (without Telmisartan and Hydrochlorothiazide), placebo with

Telmisartan and Placebo with Hydrochlorothiazide same equivalent to one tablet and transfer

in the dissolution vessel. Run the Dissolution as per the set parameters. Withdraw about 10 ml

of the sample after 60 minutes. Filter through on line SS filter, discarding first 2-3 ml of

filtrate inject the filtrate into the HPLC. (Only for validation)

Evaluation of System suitability:

Inject the Telmisartan Standard five times; the relative standard deviation of five replicate

injections should not be more than 2.0%. The USP tailing factor for Telmisartan and

Hydrochlorothiazide peak should not be more than 2.0. The USP plates for Telmisartan and

Hydrochlorothiazide peak should not be less than 2000.

Procedure:

Inject equal volumes of Blank (diluent), Standard (5 replicate) and sample solutions.

Calculation:

Calculate the amount of Telmisartan present in the tablets as per give formula.

For 80 mg

AT WS 10 900 P % Release = -------- x --------- x ------ x --------- x -------- AS 50 200 1 Tab LC

For 40 mg


CHAPTER – 6


Calculate the amount of Hydrochlorothiazide present in the tablets as per give formula.

For 12.5 mg


Where,

AT = Area of peak due to Active Ingredient sample preparation.

AS = Area of peak due to Active Ingredient in standard preparation.

WS = Weight of Active Ingredient standard in mg.

LC = Label claim of Active Ingredient per tablet in mg.

P = Potency of Active Ingredient standard on as is basis.

CHAPTER – 6


6.4 VALIDATION OF THE DEVELOPED METHOD 6.4.1 Validation parameters and acceptance criteria The Table 6.4.1.1 summarizes the validation acceptance criteria along with the obtained results.

Table 6.4.1.1: Validation Summary

Sr.No. Parameters Acceptance criteria Result obtained

1.0

System suitability

% RSD for Standard

solution

USP Tailing

USP Plate count

NMT 2.0%

NMT 2.0

NLT 2000.

Telmisartan HCTZ

0.08

1.01

3602

0.12

1.06

5155

2.0

2.1

Specificity

Identification

Results should be

comparable with

respect to the retention

time.

Retention time (min)

Std-8.281 Std-1.580

Sample-

8.274 Sample-1.578

2.2

Interference

No interference from

blank and placebo to

main component.

Complies

2.3

Peak purity

Purity angle should be

less than purity

threshold.

Standard peak should

be pure for working

concentration level.

Sample Purity

angle

Purity

Threshold

Telmisartan

Standard 0.056 1.060

Sample 0.072 1.078

HCTZ

Standard 0.168 1.097

Sample 0.188 1.117

CHAPTER – 6


Table 6.4.1.1: Validation Summary (Continued)

Sr. No. Parameters Acceptance criteria Result obtained

3.0 Linearity

Response should be Linear Response is linear

Correlation coefficient should not

be less than 0.999.

Telmisartan HCTZ

80 mg 40 mg

1.0000 1.0000 0.9998

% Limit of Y- Intercept should be

within ± 5.0% of the

corresponding

Y-co-ordinate of the working

level.

-0.39 0.34 0.76

4.0 Accuracy (Recovery) Mean recovery should be in the

range of 95.0%- 105.0%.

Level % % Mean Recovery

Telmisartan

80 mg 40 mg

10 97.3 96.5

50 98.7 99.2

100 99.3 98.4

150 101.2 99.2

HCTZ

10 100.1 96.0

50 98.7 97.2

100 103.7 103.1

150 100.8 98.4

CHAPTER – 6



Sr. No. Parameters Acceptance

criteria Result obtained

5.0

5.1

System Precision

% RSD for Standard

solution

USP Tailing

USP Plate count

NMT 2.0%

NMT 2.0

NLT 2000

Telmisartan HCTZ

80 mg 40 mg 80 mg 40 mg

0.08

1.01

3602

0.19

0.99

3621

0.12

1.06

5155

0.14

1.07

5432

Method Precision % RSD

of six determination NMT 5.0%. 1.14 1.88 1.39 1.62

5.2

Intermediate Precision

(Ruggedness)

% RSD for Standard

solution

NMT 2.0%

0.30

0.48

0.45

0.59

USP Tailing NMT 2.0 1.03 1.00 1.04 1.04

USP Plate count

NLT 2500.

3108

3123

5532

5590

RSD for % release NMT 5.0%. 2.49 1.55 1.38 1.59

Difference for pooled

result ( Analyst-I and II )

The difference in

the mean should

not be more than

5.

2.0 5.0 1.0 0.0

CHAPTER – 6



Sr. No. Parameters Acceptance criteria Result obtained

6.0

Stability in

analytical

solution

The difference should not be

more than 5.

Telmisartan HCTZ

Sample stable

for at least 24

hours at 25°C

Sample stable

for at least 24

hours at 25°C

7.0 Filter

compatibility

The difference between

centrifuged sample and filtered

sample should not be more

than 5.

complies complies

8.0

Robustness

Change in Flow

rate

(± 0.2 ml/min)

No significant change should

be in System suitability

parameters.

% RSD should be less than

5%.

No significant change.

Compiles

Change in

wavelength

(± 5nm)



parameters.


5%.


Compiles

Column oven

temperature (±

5°C)



parameters.


5%.


Compiles

Change in Mobile

phase

composition

(± 2 % absolute)



parameters.


5%.


Compiles

CHAPTER – 6


Change in Buffer

pH

(± 0.2)



parameters.


5%.


Compiles

Change in Speed

of rotation. (± 4%

rpm)



parameters.


5%.


Compiles

6.4.2 System suitability:

Single injection of Blank (Diluent) and five replicate Standard solution were made on the

system. The data obtained is summarized in Table 6.4.2.1. The data demonstrate that the

system suitability is within the acceptance criteria, thus the system is suitable

Table 6.4.2.1: System suitability

Standard solution


USP Tailing 1.01 1.06

USP Plates 3602 5155

Area

Standard solution

1590248 510817

1588324 511411

1589088 511427

1590342 510766

1587588 509953

Mean 1589118 510875

SD 1198.68 603.999

%RSD 0.08 0.12

6.4.3 Specificity:

The Specificity study included Identification of the main peak, Interference study and Peak

Purity

CHAPTER – 6


Injections of Blank and standard solutions were made as directed in the method; sample solution

and placebo preparation were made and injected into the HPLC. The data obtained is

summarized in Table 6.4.3.1. Purity angle is less than purity threshold for all components. The

data demonstrate that there is no interference in blank and placebo with Telmisartan and

Hydrochlorothiazide peaks.

Table 6.4.3.1: Specificity (Identification and Interference)

Component Retention

time (min)

USP

Plates

USP

Tailing

Purity

angle

Purity

threshold

Standard Solution

Telmisartan 8.281 3630 1.01 0.056 1.060

Hydrochlorothiazide 1.580 5348 0.99 0.168 1.097

Sample Solution Telmisartan 8.274 3648 1.01 0.074 1.086

Hydrochlorothiazide 1.578 5711 0.98 0.188 1.117

Purity angle is less than purity threshold for all components.

Chromatograms of Blank (diluent), Placebo without Telmisartan and Hydrochlorothiazide,

Placebo with Telmisartan, Placebo with Hydrochlorothiazide, Standard solution and Sample

solution are given below under figure 6.4.3.1, 6.4.3.2, 6.4.3.3, 6.4.3.4, 6.4.3.5 and 6.4.3.6

respectively

Figure-6.4.3.1: Chromatogram of Blank.

CHAPTER – 6


Figure-6.4.3.2: Chromatogram of Placebo without Telmisartan and Hydrochlorothiazide.

Figure-6.4.3.3: Chromatogram of Placebo with Telmisartan.

CHAPTER – 6


Figure-6.4.3.4: Chromatogram of Placebo with Hydrochlorothiazide

Figure-6.4.3.5: Chromatogram of Standard solution.

CHAPTER – 6


Figure-6.4.3.6: Chromatogram of Control Sample solution.

6.4.4 Linearity and Range:

The Linearity of response was determined by preparing different concentrations of standard

stock solution ranging from 10% to 150% of the working concentration of tablets (80/12.5

mg and 40/12.5 mg). The data summarized in Table 6.4.4.1, 6.4.4.2 and 6.4.4.2. The data

shows that the response is found to be linear for all constituents; Correlation coefficient is

more than 0.999. The Y-intercept is also within the set criterion

Table 6.4.4.1: Linearity of Telmisartan (80 mg Tablets)

Level Concentration

(%)

Response

1 2 Mean

1 10.01 159520 158085 158803

2 50.04 795499 794949 795224

3 55.60 883971 882956 883464

4 66.72 1061820 1058702 1060261

5 77.84 1230513 1231606 1231060

6 100.08 1596956 1597363 1597160

7 133.44 2129389 2128318 2128854

8 155.68 2482805 2483224 2483015

CORRELATION COEFFICIENT (r) 1.0000

SLOPE 15973

Y-INTERCEPT -4423

MEDIAN (AREA) 1145660

% LIMIT OF Y-INTERCEPT ± 5% OF MEDIAN -0.39

CHAPTER – 6


Table 6.4.4.2: Linearity of Telmisartan (40 mg Tablets)

Level Concentration

(%)

Response

1 2 Mean

1 10.01 79262 78778 79020

2 44.48 359804 358912 359359

3 55.60 437282 437910 437596

4 66.72 532535 530750 531643

5 100.08 795499 794949 795224

6 111.20 883971 882956 883464

7 133.44 1061820 1058702 1060261

8 155.68 1230513 1231606 1231060


SLOPE 7914

Y-INTERCEPT 2277


% LIMIT OF Y-INTERCEPT ± 5% OF MEDIAN 0.34

Table 6.4.4.3: Linearity of Hydrochlorothiazide.

Level Concentration

(%)

Response

1 2 Mean

1 11.30 53956 53803 53884

2 56.51 269135 269157 269146

3 98.90 467804 467674 467739

4 113.02 524989 524755 524872

5 127.15 584189 584178 584184

6 141.28 659175 659528 659352

7 169.54 790175 791378 790777


SLOPE 4629

Y-INTERCEPT 4004


% LIMIT OF Y-INTERCEPT ± 5% OF MEDIAN 0.76

CHAPTER – 6


The graphical depiction is included in Figures 6.4.4.1, 6.4.4.2, 6.4.4.3

Figure-6.4.4.1: Linearity for Telmisartan (80 mg Tablets).

Figure-6.4.4.2: Linearity for Telmisartan (40 mg Tablets).

CHAPTER – 6


Figure-6.4.4.3: Linearity for Hydrochlorothiazide.

CHAPTER – 6


6.4.5 Accuracy:

The standard solution was spiked into the placebo at four different levels, 10%,50%, 100%

and 150% from three different standard stock solutions and each level in duplicate were

injected. This was performed for both strengths and the results are summarized. From the

amount added and the amount found, the percentage recovery was calculated. The mean

recovery was calculated. The results obtained were summarized from Table 6.4.5.1 to

6.4.5.6. The data shows that the percentage mean recovery at each level is within the

acceptance criteria.

Table 6.4.5.1: % Recovery for Telmisartan (For 80/12.5mg Tablets)

Level

%

Response

% Recovery

Mean recovery

%

10 %

160623 95.3

97.3 159139 96.5

158146 100.0

50 %

778564 99.0

98.7 777928 98.1

782521 99.0

100 %

1577903 99.8

99.3 1569893 98.9

1570228 99.1

150 %

2379112 100.3

101.2 2405902 101.8

2412083 101.6

CHAPTER – 6


Table 6.4.5.2: % Recovery Hydrochlorothiazide (For 80/12.5mg Tablets)

Level

%

Response

% Recovery

Mean recovery

%

10 %

49707 97.2

100.1 51792 100.7

52829 102.5

50 %

252342 98.7

98.7 253555 98.6

254865 98.9

100 %

529097 103.4

103.7 532293 103.5

536431 104.1

150 %

778896 101.5

100.8 770861 99.9

781205 101.0

Table 6.4.5.3: % Recovery for Telmisartan (For 40/12.5mg Tablets)

Level

%

Response

% Recovery Mean recovery

%

10 %

76901 97.0

96.5 80293 97.2

80715 95.5

50 %

399263 99.3

99.2 397340 97.8

402408 100.6

100 %

780794 97.5

98.4 789970 98.4

788937 99.2

150 %

1187221 99.3

99.2 1182715 99.2

1174468 99.0

CHAPTER – 6


Table 6.4.5.4: % Recovery Hydrochlorothiazide (For 40/12.5mg Tablets)

Level

%

Response

% Recovery Mean recovery

%

10 %

48860 95.2

96.0 50036 96.9

49655 96.0

50 %

246541 96.1

97.2 250312 97.0

254765 98.5

100 %

522466 101.8

103.1 532310 103.1

539995 104.4

150 %

759734 98.7

98.4 758767 98.0

765570 98.7

CHAPTER – 6


6.4.6 Precision

6.4.6.1 System Precision:

Single injection of Blank (Diluent) and five replicate injections of standard solution were

made on the system. Please refer to Table 6.4.2.1 for system suitability of 80-12.5mg

strength. Table 6.4.6.1.1 depicts the system suitability for 40-12.5 strength. All the data were

acceptable as per the system suitability requirements.

Table 6.4.6.1.1: System precision (For 40/12.5 mg Tablets)

Standard solution




Area

Standard solution

794947 503794

793062 504032

793848 504781

797008 504752

795511 503147

Mean 794875 504101

SD 1524.88 688.35

%RSD 0.19 0.14

CHAPTER – 6


6.4.6.2 Method Precision:

Six independent sample solutions were prepared and injected on the HPLC. The data

obtained is summarized in Table 6.4.6.2.1 and 6.4.6.2.2 The data shows that % RSD is

within the acceptance criteria.

Table 6.4.6.2.1: Method precision (80/12.5 mg Tablets)

Tablet No. % Release


1 91 100

2 89 99

3 91 99

4 90 98

5 91 96

6 92 98

Mean 91 98

SD 1.033 1.366

% RSD 1.14 1.39

Table 6.4.6.2.2: Method precision (40/12.5 mg Tablets)



1 93 103

2 92 100

3 90 99

4 94 102

5 95 103

6 92 101

Mean 93 101

SD 1.751 1.633

% RSD 1.88 1.62

CHAPTER – 6


6.4.6.3 Intermediate Precision (Ruggedness):

Same procedure of system precision and method precision was followed by another Analyst on

different instrument and on different day. The data demonstrate that the system complied with

system suitability requirements. The data obtained from Analyst-II are summarized from Table

6.4.6.3.1 to 6.4.6.3.4. The data shows that percentage RSD is within the acceptance criteria for

system suitability as well as for intermediate precision.

Table 6.4.6.3.1: Intermediate precision – System Suitability (For 80/12.5 mg Tablets)

Standard solution




Area

Standard solution

1663210 526817

1665038 527459

1665648 527090

1671229 531085

1657249 524510

Mean 1664475 527392

SD 5028.039 2364.87

%RSD 0.30 0.45

CHAPTER – 6


Table 6.4.6.3.2: Intermediate precision – System Suitability (For 40/12.5 mg Tablets)

Standard solution




Area

Standard solution

853613 513824

861737 519757

860066 518801

862997 521692

863898 520646

Mean 860462 518944

SD 4090.34 3055.492

%RSD 0.48 0.59

Table 6.4.6.3.3: Ruggedness (80/12.5 mg Tablets)



1 91 101

2 96 99

3 93 98

4 91 101

5 96 99

6 92 98

Mean 93 99

SD 2.317 1.366

% RSD 2.49 1.38

CHAPTER – 6


Table 6.4.6.3.4: Ruggedness (40/12.5 mg Tablets)



1 88 101

2 90 102

3 87 99

4 88 101

5 90 103

6 87 99

Mean 88 101

SD 1.366 1.602

% RSD 1.55 1.59

The pooled data obtained from Analyst-I and Analyst-II is summarized in Table 6.4.6.3.5 and

6.4.6.3.6. The data shows that % difference is not more than 5.

Table 6.4.6.3.5: Pooled data (80/12.5 mg Tablets)

Analyst % Release


I

91 100

89 99

91 99

90 98

91 96

92 98

Mean 91 98

II

91 101

96 99

93 98

91 101

96 99

92 98

Mean 93 99

% Difference between

two means 2.0 1.0

CHAPTER – 6


Table 6.4.6.3.6: Pooled data (40/12.5 mg Tablets)

Analyst % Release


I

93 103

92 100

90 99

94 102

95 103

92 101

Mean 93 101

II

88 101

90 102

87 99

88 101

90 103

87 99

Mean 88 101

% Difference between

two means 5.0 0.0

6.4.7 Stability in Analytical solution:

The Sample solution was kept at sample temperature for 24 hours were injected on to the HPLC

time to time. The data obtained are summarized in Table 6.4.7.1 and 6.4.7.2. The data shows that

for Telmisartan, both the sample and standard solution were stable for (at least) upto 24 hours at

25°C.

The observation was similar for Hydrochlorothiazide.

CHAPTER – 6


Table 6.4.7.1: Stability in analytical solution (Telmisartan)

Time Standard

Area

Cumulative RSD

of Standard area

%

Release

Cumulative

%

Difference

Initial (control) 1588324 - 91 - 3 hrs 1589401 0.05 90 1

6 hrs 1594321 0.27 91 0 9 hrs 1589904 0.07 90 1

12 hrs 1593050 0.21 91 0 20 hrs 1593050 0.21 90 1 24hrs 1601447 0.58 90 1

Table 6.4.7.2: Stability in analytical solution (Hydrochlorothiazide)

Time Standard

Area

Cumulative RSD of

Standard area

%

Release

Cumulative

%

Difference

Initial (control) 511411 - 100 - 3 hrs 510253 0.16 101 1

6 hrs 508835 0.36 101 1 9 hrs 507830 0.50 101 1

12 hrs 505523 0.82 101 1 20 hrs 503509 1.10 99 1 24hrs 498312 1.83 98 2

6.4.8 Filter compatibility:

The Sample solution was centrifuged and used as control for this study. Samples filtered through

different filter were also injected on to the HPLC.

The data shows that % difference is not more than 5. Thus all filters tested were compatible

with the sample. The data obtained are summarized in Table 6.4.8.1.

CHAPTER – 6


Table 6.4.8.1: Filter compatibility

Filter


% Release Cumulative

% Difference

% Release Cumulative

% Difference

Centrifuged 95 - 102 -

Glass filter 95 0 102 0

On line SS filter 91 4 101 1

Nylon filter 93 2 101 1

Teflon filter 95 0 102 0

PVDF 97 2 102 0

6.4.9 Robustness:

The changes in system suitability parameters and results, when deliberate controlled changes

were made to the method, were studied in robustness. No significant changes in system

suitability parameters or results were observed during robustness study proving the method to be

considerable robust The data obtained are summarized in Table 6.4.9.1 and 6.4.9.2.

Table 6.4.9.1: Robustness (Telmisartan).

Changes in

parameters Values

Retention

time of

Telmisartan

USP

Plates

USP

Tailing

% RSD of

standard

area % R

elea

se

%

Dif

fere

nce

Control As per

method 8.269 3602 1.01 0.08 91 -

Flow rate (ml/min) 1.8 9.193 3722 1.01 0.17 90 1

2.2 7.527 3293 1.01 0.23 91 0

Wavelength (nm) 265 8.281 3611 1.01 0.05 91 0

275 8.281 3579 1.00 0.18 90 1 Mobile phase

composition

(Buffer:

ACN+MeOH)

78:22 5.867 3706 1.02 0.06 93 2

82:18 8.501 3330 1.04 0.05 93 2

Column

temperature

30°C 8.130 3408 1.05 0.12 93 2

40°C 7.036 3616 1.00 0.07 93 2

Buffer pH 2.8 7.791 2923 1.05 0.42 93 2

3.2 6.78 2854 1.03 0.03 93 2 Speed of rotation -

RPM

72 8.269 3602 1.01 0.08 91 0

78 8.269 3602 1.01 0.08 91 0

CHAPTER – 6


Table 6.4.9.2: Robustness (Hydrochlorothiazide).

Changes in

parameters Values

Retention

time of

HCTZ

USP

Plates

USP

Tailing

% RSD

of

standard

area % R

elea

se

%

Dif

fere

nce

Control As per

method 1.567 5155 1.06 0.12 100 -

Flow rate

(ml/min)

1.8 1.752 5651 1.06 0.05 100 0

2.2 1.437 4933 1.08 0.22 100 0

Wavelength (nm) 265 1.580 5320 0.99 0.18 100 0

275 1.580 5310 0.99 0.09 100 0 Mobile phase

composition

(Buffer:

ACN+MeOH)

78:22 1.490 5646 1.10 0.10 100 0

82:18 1.601 5075 1.06 0.06 100 0

Column

temperature

30°C 1.607 5136 1.08 0.18 99 1

40°C 1.490 5563 1.10 0.15 99 1

Buffer pH 2.8 1.553 5373 1.04 0.40 100 0

3.2 1.512 5274 1.04 0.06 100 0

Speed of rotation 72 1.567 5155 1.06 0.12 99 1

78 1.567 5155 1.06 0.12 98 2

6.4.10 Conclusions

The method has been shown to be specific for Telmisartan and Hydrochlorothiazide tablets.

The method has been shown to be Linear, precise and accurate across the suitable

analytical range and stability indicating. Solution has been shown to be stable for at least 24 hours when stored at 25°C.

The method has been shown to be robust towards deliberate minor changes in the

method parameters of both HPLC and Dissolution. The method can be used in quality control laboratory for release of production batches.

chapter – 6 simultaneous determination of drug...

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